Fluorescent chemical compounds having high selectivity for double stranded DNA, and methods for their use

ABSTRACT

Chemical compounds having a high selectivity for double stranded DNA over RNA and single stranded DNA are disclosed. The chemical compounds are stains that become fluorescent upon illumination and interaction with double stranded DNA, but exhibit reduced or no fluorescence in the absence of double stranded DNA. The compounds can be used in a variety of biological applications to qualitatively or quantitatively assay DNA, even in the presence of RNA.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 11/432,814, filed May 11, 2006 (now U.S. Pat. No. 7,598,390),which claims priority to U.S. Provisional Patent Application Ser. No.60/680,243, filed May 11, 2005, the contents of which are herebyincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to stains that become fluorescent upon interactionwith DNA. In particular, stains that exhibit higher fluorescence whencontacted with double stranded DNA than when contacted with RNA and/orsingle stranded DNA, as well as various uses for the stains aredisclosed.

DESCRIPTION OF RELATED ART

Stains and dyes are commonly used in chemical, biotechnological, andbiomedical research. These two types of compounds are different in theirproperties, and in their intended uses.

Stains are chemical compounds that exhibit a detectable response whencontacted with a particular target. In the absence of the target, astain does not exhibit the detectable response. These properties makestains valuable in the detection of the presence or absence of aparticular target in a sample. The detectable response can bequalitative or quantitative, depending on the compound, target, andassay parameters.

In comparison, dyes exhibit a detectable response regardless of thepresence or absence of another material. Dyes are therefore useful tolabel a target. For example, an antibody can be labeled with afluorescent dye molecule. The localization of the antibody in a cell ortissue can be monitored by fluorescence.

The detection and quantitation of DNA is a very common task inbiotechnological research. Early chemical stains such as ethidiumbromide are effective at staining DNA, but also stain RNA. DNA and RNAare often obtained together when isolated from natural sources. Stainsthat are not selective for DNA make quantitation of the isolated DNAdifficult, requiring a purification step to be performed prior toquantitation. Stains find use in applications such as gelelectrophoresis, PCR, real time PCR quantitation, DNA solutionquantitation, microarrays, and RT-PCR.

Multiple nucleic acid stains are commercially available. The followingis a representative listing of these materials.

Ethidium bromide is the most widely used nucleic acid stain, and iscommercially available from a wide array of suppliers. Ethidium bromideis mutagenic, and its use requires significant care from the user toavoid contact with staining solutions.

PicoGreen is a stain selective for double stranded DNA (commerciallyavailable from Molecular Probes, Inc. (Eugene, Oreg.) since 1994).PicoGreen shows a greater than 1000 fold fluorescence enhancement uponbinding to double stranded DNA, and much less enhancement upon bindingto single stranded DNA or RNA.

OliGreen is a stain useful for the quantitation of single stranded DNAsuch as synthetic oligonucleotides. OliGreen has been commerciallyavailable from Molecular Probes, Inc. (Eugene, Oreg.) since 1994.Quantitation with OliGreen is about 10,000 times more sensitive thanquantitation with UV absorbance methods, and at least 500 times moresensitive than detecting oligonucleotides on electrophoretic gelsstained with ethidium bromide. This type of material is described inU.S. Pat. Nos. 5,436,134 and 5,658,751; Australian Patent Nos. 676,317and 714,890; Canadian Patent No. 2,133,765; and European Patent Nos.0,675,924 and 0,740,689.

RiboGreen is a stain that is useful for the quantitation of RNA insolution. RiboGreen has been commercially available from MolecularProbes, Inc. (Eugene, Oreg.) since 1997. This type of material isdescribed in U.S. Pat. Nos. 5,658,751 and 5,863,753; Australian PatentNo. 714,890; and European Patent No. 0,740,689.

SYBR Green I is stain selective for DNA (commercially available fromMolecular Probes, Inc. (Eugene, Oreg.) since 1993). SYBR Green I has afluorescence enhancement upon binding to DNA at least 10 fold greaterthan that of ethidium bromide, and a fluorescence quantum yield overfive times greater than ethidium bromide (about 0.8 as compared to about0.15). This type of material is described in U.S. Pat. Nos. 5,436,134and 5,658,751; Australian Patent Nos. 676,317 and 714,890; CanadianPatent No. 2,133,765; and European Patent Nos. 0,675,924 and 0,740,689.

SYBR Safe is a nucleic acid stain that is at least twice as sensitive asethidium bromide, yet exhibits reduced mutagenicity. SYBR Safe has beencommercially available from Molecular Probes, Inc. (Eugene, Oreg.) since2003. This type of material is described in U.S. Pat. Nos. 4,883,867,4,957,870, 5,436,134, and 5,658,751; Australian Patent Nos. 676,317 and714,890; Canadian Patent No. 2,133,765; and European Patent Nos.0,675,924 and 0,740,689.

Hoechst 33258 (CAS 23491-45-4; Phenol,4-[5-(4-methyl-1-piperazinyl)[2,5′-bi-1H-benzimidazol]-2′-yl]-,trihydrochloride) is a nuclear counterstain that emits blue fluorescencewhen bound to dsDNA. Hoechst 33258 has been commercially available fromMolecular Probes, Inc. (Eugene, Oreg.) since 1992.

Dimeric cyanines TOTO-1, YOYO-1, and YO-PRO-1 are useful for themeasurement of double stranded DNA, single stranded DNA, and RNA insolution. TOTO-1, YOYO-1, and YO-PRO-1 have been commercially availablefrom Molecular Probes, Inc. (Eugene, Oreg.) since 1992. These types ofmaterials are described in U.S. Pat. Nos. 5,321,130 and 5,582,977;Canadian Patent No. 2,119,126; and European Patent No. 0,605,655 B1.

Unsymmetrical cyanine dyes having similar spectral properties tointercalating cyanine dyes, but binding in the minor groove of DNA werereported in 2003 (Karlsson, H. J. et al., Nucleic Acids Res. 31(21):6227-6234 (2003)). Compounds BEBO, BETO, and BOXTO were shown, andcharacterized using a variety of spectral measurements. Fluorescencequantum yield increased upon binding to DNA, but RNA binding resultswere not shown.

Despite the materials and methods that are currently available, therestill exists a need for stains that are selective for double strandedDNA in the presence of RNA, single stranded DNA, or other biologicalmaterials.

SUMMARY OF THE INVENTION

Compounds are disclosed having high selectivity for double stranded DNAover RNA and single stranded DNA. The compounds act as fluorescentstains, where they exhibit fluorescent properties when illuminated inthe presence of double stranded DNA, but exhibit reduced or nofluorescence in the presence of RNA, single stranded DNA, or in theabsence of nucleic acids entirely. The compounds can contain the corestructure of Compound (1A) or Compound (1B).

Also disclosed are methods for the preparation of the compounds, andmethods for their use in detecting the presence or absence of doublestranded DNA in a sample. The selectivity of the compounds for doublestranded DNA over RNA and single stranded DNA enables detection ofdouble stranded DNA in samples containing RNA and/or single strandedDNA.

DETAILED DESCRIPTION OF THE INVENTION

While compositions and methods are described in terms of “comprising”various components or steps (interpreted as meaning “including, but notlimited to”), the compositions and methods can also “consist essentiallyof” or “consist of” the various components and steps, such terminologyshould be interpreted as defining essentially closed-member groups.

Compounds

A first embodiment of the invention is directed towards chemicalcompounds. The chemical compounds can be neutrally charged, positivelycharged, or negatively charged. When positively or negatively charged,the compound can include one or more counterions.

One embodiment of the invention is directed towards chemical compoundscontaining the core structure of Compound (1A).

The double bond(s) in the center of Compound (1A) can be in either cisor trans configuration. For example, if X is nitrogen, the two nitrogenscan be oriented on the same side of the central double bond (cis) or canbe oriented across the central double bond (trans). Mixtures of bothconfigurations are also possible in a sample of a particular compound.

The value n can be any non-negative integer. For example, n can be zero,1, 2, 3, 4, 5, 6, 7, 8, and so on.

X can be oxygen or sulfur.

Groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, and R¹¹ canindependently comprise or be hydrogen (H), hydroxyl group (OH), alkoxygroup (OR), thiol (SH), thioalkyl (SR), thioaryl (SAr), halogen (X),alkyl group, alkenyl group, alkynyl group, aromatic group, amine group(primary NH₂, secondary NHR, tertiary NR′R″, or tertiary NR′₂), areactive group, or a mixed group having combinations of two or more ofthese groups (for example, an alkyl group having thiol and aminosubstituents, an alkoxy group having amino substituents, and so on).Alternatively, one or more of these groups can be a linker group forcovalently attaching Compound (1A) to another compound. Linking thelinker group to another compound would afford a conjugate of Compound(1A).

In one embodiment, at least one of R¹, R², R³, and R⁴ comprises or is anaromatic group or an alkynyl group.

In one embodiment, R⁹ comprises or is an aromatic group, alkyl-aromatic,or an alkynyl group.

In one embodiment, R¹⁰ comprises or is an amine group.

In one embodiment, at least one of R¹, R², R³, and R⁴ comprises or is anaromatic group or an alkynyl group; and R⁹ comprises or is an aromaticgroup, alkyl-aromatic, or an alkynyl group.

In one embodiment, at least one of R¹, R², R³, and R⁴ comprises or is anaromatic group or an alkynyl group; R⁹ comprises or is an aromaticgroup, alkyl-aromatic group, or an alkynyl group; and R¹⁰ comprises oris an amine group.

Group R¹² can be an alkyl group such as a C₁-C₈ alkyl group. The C₁-C₈alkyl group can be a straight chain, branched, or cycloalkyl group.Examples of the C₁-C₈ alkyl group include methyl, ethyl, 1-propyl,2-propyl, 1-butyl, 2-butyl, 1-pentyl, 1-hexyl, 1-heptyl, and 1-octyl. Ina presently preferred embodiment, the C₁-C₈ alkyl group is a methylgroup.

When Compound (1A) is a cationic or anionic structure, it can furthercomprise one or more appropriate counterions. For example, if Compound(1A) is cationic (positively charged), it can further comprise anionssuch as chloride, bromide, iodide, sulfate, and carbonate counterions.Alternatively, if Compound (1A) is anionic (negatively charged), it canfurther comprise cations such as potassium, sodium, ammonium, magnesium,and calcium.

An additional embodiment of the invention is directed towards chemicalcompounds containing the core structure of Compound (1B).

The double bond(s) in the center of Compound (1B) can be in either cisor trans configuration. For example, if X is nitrogen, the two nitrogenscan be oriented on the same side of the central double bond (cis) or canbe oriented across the central double bond (trans). Mixtures of bothconfigurations are also possible in a sample of a particular compound.

The value n can be any non-negative integer. For example, n can be zero,1, 2, 3, 4, 5, 6, 7, 8, and so on.

X can be oxygen or sulfur.

Groups R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, and R²¹ can independentlycomprise or be hydrogen (H), hydroxyl group (OH), alkoxy group (OR),thiol (SH), thioalkyl (SR), thioaryl (SAr), halogen (X), alkyl group,alkenyl group, alkynyl group, aromatic group, amine group (primary NH₂,secondary NHR, tertiary NR′R″, or tertiary NR′₂), a reactive group, or amixed group having combinations of two or more of these groups (forexample, an alkyl group having thiol and amino substituents, an alkoxygroup having amino substituents, and so on). Alternatively, one or moreof these groups can be a linker group for covalently attaching Compound(1B) to another compound. Linking the linker group to another compoundwould afford a conjugate of Compound (1B).

In one embodiment, at least one of R¹³, R¹⁴, R¹⁵, and R¹⁶ comprises oris an aromatic group or an alkynyl group.

In one embodiment, R¹⁹ comprises or is an aromatic group,alkyl-aromatic, or an alkynyl group.

In one embodiment, R²⁰ comprises or is an amine group.

In one embodiment, at least one of R¹³, R¹⁴, R¹⁵, and R¹⁶ comprises oris an aromatic group or an alkynyl group; and R¹⁹ comprises or is anaromatic group, alkyl -aromatic, or an alkynyl group.

In one embodiment, at least one of R¹³, R¹⁴, R¹⁵, and R¹⁶ comprises oris an aromatic group or an alkynyl group; R¹⁹ comprises or is anaromatic group, alkyl-aromatic, or an alkynyl group; and R²⁰ comprisesor is an amine group.

Group R²² can be an alkyl group such as a C₁-C₈ alkyl group. The C₁-C₈alkyl group can be a straight chain, branched, or cycloalkyl group.Examples of the C₁-C₈ alkyl group include methyl, ethyl, 1-propyl,2-propyl, 1-butyl, 2-butyl, 1-pentyl, 1-hexyl, 1-heptyl, and 1-octyl. Ina presently preferred embodiment, the C₁-C₈ alkyl group is a methylgroup.

When Compound (1B) is a cationic or anionic structure, it can furthercomprise one or more appropriate counterions. For example, if Compound(1A) is cationic (positively charged), it can further comprise anionssuch as chloride, bromide, iodide, sulfate, and carbonate counterions.Alternatively, if Compound (1A) is anionic (negatively charged), it canfurther comprise cations such as potassium, sodium, ammonium, magnesium,and calcium.

Substituents

The alkoxy group can generally be any unsubstituted alkoxy group orsubstituted alkoxy group. Unsubstituted alkoxy groups contain an oxygenconnected to an alkyl group. Substituted alkoxy groups contain an oxygenconnected to a substituted alkyl group. Examples of unsubstituted alkoxygroups include methoxy (OCH₃), ethoxy (OCH₂CH₃), propoxy (OCH₂CH₂CH₃),and higher straight chain alkoxy groups. Unsubstituted alkoxy groupsalso include branched or cyclic alkoxy groups. Examples of branchedalkoxy groups include 2-propoxy (OCH(CH₃)₂), 2-butoxy (OCH(CH₃)CH₂CH₃),and higher branched alkoxy groups. Cyclic alkoxy groups have an oxygenconnected to a cyclic group. Examples of cyclic alkoxy groups includecyclopropoxy (oxygen connected to a cyclopropane ring), cyclobutoxy(oxygen connected to a cyclobutane ring), cyclopentoxy (oxygen connectedto a cyclopentane ring), cyclohexoxy (oxygen connected to a cyclohexanering), and higher cyclic alkoxy groups.

The halogen can generally be any halogen. Halogen groups include chloro,fluoro, bromo, and iodo groups.

Alkyl groups can generally be any unsubstituted or substituted alkylgroup. Unsubstituted alkyl groups contain only carbon and hydrogenatoms. Substituted alkyl groups can contain one or more non-carbon andnon-hydrogen atoms such as oxygen, nitrogen, sulfur, halogens, andphosphorous.

Alkenyl groups can generally be any alkenyl group containing at leastone carbon-carbon double bond. The most simple alkenyl group is a vinylgroup (—CH═CH₂). Higher alkenyl groups include 1-propenyl (—CH═CH₂CH₃),1-butenyl (—CH═CH₂CH₂CH₃), 2-butenyl (—CH₂—CH═CHCH₃), and 3-butenyl(—CH₂CH₂CH═CH₂). Substituted alkenyl groups can contain one or morenon-carbon and non-hydrogen atoms such as oxygen, nitrogen, sulfur,halogens, and phosphorous.

Alkynyl groups can generally be any alkynyl group containing at leastone carbon-carbon triple bond. The most simple alkynyl group is anethynyl group (—CCH). Higher alkynyl groups include propargyl (—CH₂CCH),2-butynyl (—CH₂CCCH₃), and 3-butynyl (—CH₂CH₂CCH).

A simple example of an aryl group is a phenyl group. The aryl group canbe a simple unsubstituted aryl group containing carbon and hydrogen, orit can be a substituted aryl group. Aryl groups can include one or morearomatic rings. The aryl group can be a polycyclic aromatic hydrocarbon,or can be a heteroaryl group. Examples of aryl and heteroaryl groupsinclude phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, anthracenyl,acenaphthalenyl, acenaphthenyl, benzo[a]pyrenyl, benz[a]anthracenyl,1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2 imidazolyl,4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl,4-thiazolyl, 5-thiazolyl, 2 furyl, 3-furyl, 2-thienyl, 3-thienyl,2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4 pyrimidyl,5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, tetrazolyl,benzo[b]furanyl, benzo[b]thienyl, 2,3-dihydrobenzo[1,4]dioxin-6-yl,benzo[1,3]dioxol-5-yl, and 6-quinolyl. Heteroatoms in the heteroarylgroup can include one or more of nitrogen (N), oxygen (O), sulfur (S),and phosphorous (P).

Aryl groups can be connected to the central core structure Compound (1A)or Compound (1B), either directly by a covalent bond, or indirectlythrough one or more atoms. For example, N, O, P, or S atoms can be usedto link the aryl group to Compound (1A) or Compound (1B). Examples ofthis include phenylamino (NHC₆H₅), diphenylamino (N(C₆H₅)₂), phenoxy(OC₆H₅), and thiophenyl (SC₆H₅). Alternatively, alkyl groups can be usedto link the aryl group to Compound (1A) or Compound (1B). An example ofthis would be a benzyl group (CH₂C₆H₅; where a methylene CH₂ groupconnects the phenyl group to Compound (1A) or Compound (1B)), or astyrene group (CH═CH—C₆H₅).

Amine or amino groups can include NH₂, NHR, NR₂, and NR′R″ groups. TheR, R′, and R″ groups can be unsubstituted alkyl groups, substitutedalkyl groups, unsubstituted alkenyl groups, substituted alkenyl groups,unsubstituted alkynyl groups, substituted alkynyl groups, unsubstitutedaromatic groups, or substituted aromatic groups.

The chemical compound can comprise at least one reactive group capableof reacting with another species, such as an atom or chemical group toform a covalent bond, that is, a group that is covalently reactive undersuitable reaction conditions, and generally represents a point ofattachment for another substance, for example, a carrier molecule or asubstrate. For example, the reactive group on a disclosed compound is amoiety, such as carboxylic acid or succinimidyl ester, on the compoundsthat can chemically react with a functional group on a differentcompound to form a covalent linkage. Reactive groups generally includenucleophiles, electrophiles and photoactivatable groups.

The reactive group can be covalently attached directly to the Compound(1A) core structure, or can be covalently attached to at least one ofthe R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, and R¹¹ groups. Thereactive group can be covalently attached directly to the Compound (1B)core structure, or can be covalently attached to at least one of theR¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, and R²¹ groups

Exemplary reactive groups include, but are not limited to, olefins,acetylenes, alcohols, phenols, ethers, oxides, halides, aldehydes,ketones, carboxylic acids, esters, amines, amides, cyanates,isocyanates, thiocyanates, isothiocyanates, hydrazines, hydrazones,hydrazides, diazo groups, diazonium groups, nitro groups, nitriles,mercaptans, sulfides, disulfides, sulfoxides, sulfones, sulfonic acidgroups, sulfinic acid groups, acetals, ketals, anhydrides, sulfates,sulfenic acid groups, isonitriles, amidines, imides, imidates, nitrones,hydroxylamines, oximes, hydroxamic acid groups, thiohydroxamic acidgroups, allenes, ortho esters, sulfites, enamines, ynamines, ureas,pseudoureas, semicarbazides, carbodiimides, carbamates, imines, azides,azo groups, azoxy groups, and nitroso groups. Reactive functional groupsalso include those used to prepare bioconjugates, for example,N-hydroxysuccinimide esters, maleimides, and the like. Methods toprepare each of these functional groups are well known in the art andtheir application to or modification for a particular purpose is withinthe ability of one of skill in the art (see, for example, Sandler andKaro, eds., Organic Functional Group Preparations, Academic Press, SanDiego, 1989). Reactive groups include those shown in the followingtable.

Electrophilic Group Nucleophilic Group Resulting Covalent Linkageactivated esters* amines/anilines carboxamides acrylamides thiolsthioethers acyl azides** amines/anilines carboxamides acyl halidesamines/anilines carboxamides acyl halides alcohols/phenols esters acylnitriles alcohols/phenols esters acyl nitriles amines/anilinescarboxamides aldehydes amines/anilines imines aldehydes or ketoneshydrazines hydrazones aldehydes or ketones hydroxylamines oximes alkylhalides amines/anilines alkyl amines alkyl halides carboxylic acidsesters alkyl halides thiols thioethers alkyl halides alcohols/phenolsethers alkyl sulfonates thiols thioethers alkyl sulfonates carboxylicacids esters alkyl sulfonates alcohols/phenols ethers anhydridesalcohols/phenols esters anhydrides amines/anilines carboxamides arylhalides thiols thioethers aryl halides amines aryl amines aziridinesthiols thioethers boronates glycols boronate esters carbodiimidescarboxylic acids N-acylureas or anhydrides diazoalkanes carboxylic acidsesters epoxides thiols thioethers haloacetamides thiols thioethershaloplatinate amino platinum complex haloplatinate heterocycle platinumcomplex haloplatinate thiol platinum complex halotriazinesamines/anilines aminotriazines halotriazines alcohols/phenols triazinylethers halotriazines thiols triazinyl thioethers imido estersamines/anilines amidines isocyanates amines/anilines ureas isocyanatesalcohols/phenols urethanes isothiocyanates amines/anilines thioureasmaleimides thiols thioethers phosphoramidites alcohols phosphite esterssilyl halides alcohols silyl ethers sulfonate esters amines/anilinesalkyl amines sulfonate esters thiols thioethers sulfonate esterscarboxylic acids esters sulfonate esters alcohols ethers sulfonylhalides amines/anilines sulfonamides sulfonyl halides phenols/alcoholssulfonate esters *Activated esters, as understood in the art, generallyhave the formula COΩ, where Ω is a good leaving group (e.g.succinimidyloxy (—OC₄H₄O₂) sulfosuccinimidyloxy (—OC₄H₃O₂—SO₃H),1-oxybenzotriazolyl (—OC₆H₄N₃); or an aryloxy group or aryloxysubstituted one or more times by electron withdrawing substituents suchas nitro, fluoro, chloro, cyano, or trifluoromethyl, or combinationsthereof, used to form activated aryl esters; or a carboxylic acidactivated by a carbodiimide to form an anhydride or mixed anhydride—OCOR_(a) or —OCNR_(a)NHR_(b), where R_(a) and R_(b), which may be thesame or different, are C₁-C₆ alkyl, C₁-C₆ perfluoroalkyl, or C₁-C₆alkoxy; or cyclohexyl, 3-dimethylaminopropyl, or N-morpholinoethyl).**Acyl azides can also rearrange to isocyanates

Typically, the reactive group will react with an amine, a thiol, analcohol, an aldehyde, a ketone, or with silica. Preferably, reactivegroups react with an amine or a thiol functional group, or with silica.In one embodiment, the reactive group is an acrylamide, an activatedester of a carboxylic acid, an acyl azide, an acyl nitrile, an aldehyde,an alkyl halide, a silyl halide, an anhydride, an aniline, an arylhalide, an azide, an aziridine, a boronate, a diazoalkane, ahaloacetamide, a halotriazine, a hydrazine (including hydrazides), animido ester, an isocyanate, an isothiocyanate, a maleimide, aphosphoramidite, a reactive platinum complex, a sulfonyl halide, or athiol group. By “reactive platinum complex” is particularly meantchemically reactive platinum complexes such as described in U.S. Pat.No. 5,714,327 (issued Feb. 3, 1998).

Where the reactive group is an activated ester of a carboxylic acid,such as a succinimidyl ester of a carboxylic acid, a sulfonyl halide, atetrafluorophenyl ester, a pentafluorophenyl ester, or anisothiocyanates, the resulting compound is particularly useful forpreparing conjugates of carrier molecules such as proteins, nucleotides,oligonucleotides, or haptens. Where the reactive group is a maleimide,haloalkyl or haloacetamide (including any reactive groups disclosed inU.S. Pat. Nos. 5,362,628; 5,352,803 and 5,573,904) the resultingcompound is particularly useful for conjugation to thiol-containingsubstances. Where the reactive group is a hydrazide, the resultingcompound is particularly useful for conjugation to periodate-oxidizedcarbohydrates and glycoproteins, and in addition is an aldehyde-fixablepolar tracer for cell microinjection. Where the reactive group is asilyl halide, the resulting compound is particularly useful forconjugation to silica surfaces, particularly where the silica surface isincorporated into a fiber optic probe subsequently used for remote iondetection or quantitation.

The reactive group can be a photoactivatable group such that the groupis only converted to a reactive species after illumination with anappropriate wavelength. An appropriate wavelength is generally a UVwavelength that is less than 400 nm. This method provides for specificattachment to only the target molecules, either in solution orimmobilized on a solid or semi-solid matrix. Photoactivatable reactivegroups include, without limitation, benzophenones, aryl azides anddiazirines.

The reactive group can be a photoactivatable group, succinimidyl esterof a carboxylic acid, a haloacetamide, haloalkyl, a hydrazine, anisothiocyanate, a maleimide group, an aliphatic amine, a silyl halide, acadaverine or a psoralen. The reactive group can be a succinimidyl esterof a carboxylic acid, a maleimide, an iodoacetamide, or a silyl halide.The reactive group can be a succinimidyl ester of a carboxylic acid, asulfonyl halide, a tetrafluorophenyl ester, an iosothiocyanates or amaleimide.

The selection of a covalent linkage to attach the compound to thecarrier molecule or solid support typically depends on the chemicallyreactive group on the component to be conjugated. Examples of reactivegroups include amines, thiols, alcohols, phenols, aldehydes, ketones,phosphates, imidazoles, hydrazines, hydroxylamines, disubstitutedamines, halides, epoxides, sulfonate esters, purines, pyrimidines,carboxylic acids, or a combination of these groups. A single type ofreactive site may be available on the component (typical forpolysaccharides), or a variety of sites may occur (e.g. amines, thiols,alcohols, phenols), as is typical for proteins. A carrier molecule orsolid support may be conjugated to more than one reporter molecule,which may be the same or different, or to a substance that isadditionally modified by a hapten.

In an alternative embodiment, the present compound is covalently boundto a carrier molecule. If the compound has a reactive group, then thecarrier molecule can alternatively be linked to the compound through thereactive group. The reactive group may contain both a reactivefunctional moiety and a linker, or only the reactive functional moiety.

A variety of carrier molecules exist. Examples of carrier moleculesinclude antigens, steroids, vitamins, drugs, haptens, metabolites,toxins, environmental pollutants, amino acids, peptides, proteins,nucleic acids, nucleic acid polymers, carbohydrates, lipids, andpolymers.

The carrier molecule can comprise an amino acid, a peptide, a protein, apolysaccharide, a nucleoside, a nucleotide, an oligonucleotide, anucleic acid, a hapten, a psoralen, a drug, a hormone, a lipid, a lipidassembly, a synthetic polymer, a polymeric microparticle, a biologicalcell, a virus and combinations thereof. Alternatively, the carriermolecule can be a hapten, a nucleotide, an oligonucleotide, a nucleicacid polymer, a protein, a peptide or a polysaccharide. The carriermolecule can be an amino acid, a peptide, a protein, a polysaccharide, anucleoside, a nucleotide, an oligonucleotide, a nucleic acid, a hapten,a psoralen, a drug, a hormone, a lipid, a lipid assembly, a tyramine, asynthetic polymer, a polymeric microparticle, a biological cell,cellular components, an ion chelating moiety, an enzymatic substrate ora virus. Alternatively, the carrier molecule is an antibody or fragmentthereof, an antigen, an avidin or streptavidin, a biotin, a dextran, anantibody binding protein, a fluorescent protein, agarose, and anon-biological microparticle. The carrier molecule can be an antibody,an antibody fragment, antibody-binding proteins, avidin, streptavidin, atoxin, a lectin, or a growth factor. Examples of haptens include biotin,digoxigenin and fluorophores.

Antibody binding proteins include protein A, protein G, soluble Fcreceptor, protein L, lectins, anti-IgG, anti-IgA, anti-IgM, anti-IgD,anti-IgE or a fragment thereof.

The chemical compound can be covalently bonded to another molecule suchas an antibody, protein, peptide, polypeptide, amino acid, enzyme,nucleic acid, lipid, polysaccharide, drug, a bead, a solid support (suchas glass or plastic), and so on.

The chemical compounds preferably exhibit little or no fluorescence whenin the absence of nucleic acids. Fluorescence can be determined byilluminating the chemical compound with an appropriate wavelength, andmonitoring emitted fluorescence. The chemical compounds preferablyexhibit greater fluorescence when in the presence of DNA than when inthe presence of RNA. The fluorescence in the presence of DNA to thefluorescence in the presence of RNA is determined using a fixedconcentration of chemical compound, and a fixed concentration of DNA andRNA. Higher DNA/RNA fluorescence ratios are preferred for the detectionof DNA in the presence of RNA. The DNA/RNA ratio is preferably greaterthan about 1. More preferred ratios are greater than about 2, greaterthan about 3, greater than about 4, greater than about 5, greater thanabout 6, greater than about 7, greater than about 8, greater than about9, greater than about 10, greater than about 15, greater than about 20,greater than about 25, greater than about 30, greater than about 35,greater than about 40, greater than about 45, greater than about 50,greater than about 55, greater than about 60, greater than about 65,greater than about 70, greater than about 75, greater than about 80,greater than about 85, greater than about 90, greater than about 95,greater than about 100, greater than about 150, greater than about 200,and ranges between any two of these values.

The chemical compounds can also be characterized by their excitation andemission maxima wavelengths. For example, the excitation maximum can beabout 450 nm to about 650 nm. Excitation maxima between these values caninclude about 450 nm, about 475 nm, about 500 nm, about 525 nm, about550 nm, about 575 nm, about 600 nm, about 625 nm, about 650 nm, andranges between any two of these values. For example, the emissionmaximum can be about 500 nm to about 675 nm. Emission maxima betweenthese values can include about 500 nm, about 525 nm, about 550 nm, about575 nm, about 600 nm, about 625 nm, about 650 nm, about 675 nm, andranges between any two of these values.

Specific examples of chemical compounds include Compounds 16-35, 38-39,43-53, 55-58, 60, 62, 64-81, 83, 85-89, 91-97, 99-106, and 109-112.

Compositions

An additional embodiment of the invention is directed towardscompositions comprising one or more of the above described compounds.The compositions can comprise, consist essentially of, or consist of oneor more of the above described compounds.

The compositions can comprise one, two, three, four, or more of theabove described compounds. The compositions can further comprise asolvent. The solvent can be aqueous, non-aqueous, or a mixedaqueous/non-aqueous solvent system. Examples of solvents include water,methanol, ethanol, dimethylsulfoxide (“DMSO”), dimethylformamide(“DMF”), dimethylacetamide, and N-methylpyrrolidinone (“NMP”). Thecompositions can further comprise one or more salts or buffers.

The above described compounds can individually be present in thecomposition at a particular concentration. In one embodiment, thecompound is present in a substantially pure form without other materialspresent (sometimes referred to as “neat”). Alternatively, the compoundscan be present in a dry mixture or dissolved in a solvent or solventsystem. When dissolved, the compound can generally be present at anyconcentration. The compound can be dissolved in a concentrated solution,or in a final “working” solution. For example, a compound can be presentin a working solution at about 1 μM to about 10 μM. The concentratedsolution can have the compound at a higher concentration such as about10 μM, about 50 μM, about 100 μM, about 500 μM, about 1 mM, and rangesbetween any two of these values.

Kits

One embodiment of the invention is directed towards kits comprising oneor more of the above described compounds. The kits can comprise one,two, three, four, or more of the above described compounds. The kitpreferably comprises at least one container comprising at least one ofthe above described compounds. The kit can comprise multiple containers,such as a first container, a second container, a third container, and soon. The kit can comprise pipettes, droppers, or other sample handlingdevices. The kit can comprise a cuvette, microwell plate, or other testcontainer suitable for use in an instrument that detects emittedfluorescent energy.

The kit can comprise positive and/or negative samples. Positive samplescan comprise DNA, and/or DNA in the presence of RNA. Negative samplescan comprise RNA without DNA, or samples lacking nucleic acids entirely.The kit can comprise DNA, RNA, or both DNA and RNA.

The kit can comprise one or more additional dyes or stains. For example,the kit can contain a total nucleic acid stain. The kit can contain acell impermeant nucleic acid stain to aid in distinguishing live cellsfrom dead cells.

The kits can further comprise an instruction protocol for their use. Thekit can further comprise water, a buffer, a buffer salt, surfactants,detergents, salts, polysaccharides, or other materials commonly used inassaying biological systems. The kit can comprise solvents such asaqueous, non-aqueous, or a mixed aqueous/non-aqueous solvent systems.

Methods of Preparation

An additional embodiment of the invention is directed towards methodsfor the preparation of the above described compounds. Illustrativeexamples of these methods are described in the Examples below.

Additional embodiments of the invention include synthetic intermediates.Many synthetic intermediates are shown in the Examples section below.Examples of such intermediates include Compounds 2-15, 36-37, 40-42, 54,59, 61, 63, 82, 84, 90, and 98.

Methods of Use

An additional embodiment of the invention is directed towards methods ofusing the above described compounds.

The above described compounds can be used in methods to detect thepresence or absence of double stranded DNA in a sample. The method cancomprise providing a sample suspected of containing double stranded DNA;contacting the sample with at least one of the above described chemicalcompounds to prepare a test sample; and illuminating the test samplewith energy. The method can further comprise detecting emission ofenergy from the test sample after the illuminating step. The detectingstep can be qualitative or quantitative. The method can further comprisecalculating the concentration of double stranded DNA in the sample afterthe detecting step. The calculating step can comprise correlating theemitted fluorescent energy with the concentration of double stranded DNAin the sample.

The presence of an emitted fluorescent energy (or an increase in emittedfluorescent energy relative to a control) is indicative of the presenceof double stranded DNA in the sample, while the absence of emittedfluorescent energy (or no increase or no change relative to a control)is indicative of the absence of DNA in the sample.

The methods can also be performed on “blank” or “control” samples. Theblank or control samples can contain RNA but lack double stranded DNA,or can lack nucleic acids altogether.

The sample can generally be any type of sample. For example, the samplecan be a cell or group of cells, an organism, cell lysates, a cellculture medium, a bioreactor sample, and so on. Alternatively, thesample can be a non-biological sample. The cells can be any type ofcell, such as bacterial cells, fungal cells, insect cells, and mammaliancells. The sample can be a solid, a liquid, or a suspension. The samplecan be a biological fluid such as blood, plasma, or urine. The samplecan be a material immobilized in a gel, on a membrane, bound to a beador beads, arranged in an array, and so on. The sample can be a partiallyor fully purified nucleic acid preparation in a buffer or in water.

The contacting step can be performed at any suitable temperature, andfor any suitable length of time. Typically, the temperature will beambient or room temperature, or at an elevated temperature such as 37°C. Examples of temperatures include about 20° C., about 25° C., about30° C., about 35° C., about 37° C., about 40° C., about 42° C., andranges between any two of these values. Temperatures higher than about42° C., and temperatures lower than about 20° C. are also possible,depending on the sample tested. The length of time can generally be anylength of time suitable for detection of a change in fluorescence.Examples of lengths of time include about 10 minutes, about 20 minutes,about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes,about 70 minutes, about 80 minutes, about 90 minutes, about 100 minutes,about 110 minutes, about 120 minutes, about 180 minutes, about 240minutes, about 300 minutes, about 360 minutes, about 420 minutes, about480 minutes, about 540 minutes, about 600 minutes, and ranges betweenany two of these values. Further extended lengths of time are alsopossible, depending on the sample tested. The contacting step ispreferably performed with the test sample protected from light.

The compound or compounds can be used at generally any concentrationsuitable to produce a detectable emitted fluorescent energy signal inthe presence of double stranded DNA. Example concentration rangesinclude about 10 nM to about 1 mM. Examples of concentrations includeabout 10 nM, about 100 nM, about 1 μM, about 2 μM, about 3 μM, about 4μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10μM, about 100 μM, about 1 mM, and ranges between any two of thesevalues.

The excitation energy can be applied to the test sample in a variety ofways during the illuminating step. Suitable equipment includes hand-heldultraviolet lamps, mercury arc lamps, xenon lamps, lasers (such as argonand YAG lasers), and laser diodes. These illumination sources aretypically optically integrated into laser scanners, fluorescencemicroplate readers or standard or microfluorometers.

The detecting step can be performed by visual inspection, or by the useof a variety of instruments. Examples of such instruments include CCDcameras, video cameras, photographic film, laser scanning devices,fluorometers, photodiodes, quantum counters, epifluorescencemicroscopes, scanning microscopes, flow cytometers, fluorescencemicroplate readers, or by amplification devices such as photomultipliertubes.

The detecting step can be performed at a single point in time, can beperformed at multiple points in time, or can be performed continuously.

The methods can be used in conjunction with experimental systems such asDNA minipreps, flow cytometry, fluorescence microscopy, real time PCR,double stranded DNA quantitation, microarray hybridizations, doublestranded DNA detection in gels, and so on.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor(s) to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the scope of theinvention.

EXAMPLES Example 1 Preparation of Compound (2)

To 10 g of N-methylaniline in 200 mL acetic acid at room temperature11.3 mL bromine was added and the mixture was stirred for several hours.Volatile components were evaporated under reduced pressure and theresidue was dissolved in chloroform and washed with saturated solutionsof NaHCO₃ and Na₂S₂O₃. The crude was purified on a silica gel columnwith ethyl acetate and hexanes.

Example 2 Preparation of Compound (3)

To 3.5 g of NaH (60% by weight in dispersion oil and washed withhexanes) in 200 mL of DMF 18.5 g of Compound (2) was added, followed by6.2 mL of CS₂. The mixture was heated at 100° C. for 4 hours. Water wasadded and the solid was filtered and purified by silica gel column withethyl acetate and hexanes.

Example 3 Preparation of Compound (4)

To 9.5 g of Compound (3), 10 g of 2,6-dimethoxyphenylboronic acid and 1g of triphenylphosphine in 300 mL of isopropyl alcohol and 50 mL oftoluene was added a solution of 6 g of K₂CO₃ in 40 mL of water and 0.25g of palladium(II)acetate. The resulting mixture was heated at 100° C.for 5 hours. The solvent was removed and the residue was dissolved inCHCl₃ and washed with water. The crude product was purified on a silicagel column with ethyl acetate and hexanes.

Example 4 Preparation of Compound (5)

Compound (3) was coupled with 2,6-dimethoxyphenylboronic acid asdescribed in Example 3 (Preparation of compound (4)). This was followedby quarternization with methyl tosylate at about 130° C. for 1.5 hours.

Example 5 Preparation of Compound (6)

Compound (3) was coupled with 5-indolylboronic acid using the conditionsdescribed in Example 3 (Preparation of compound (4)). This was followedby quarternization with methyl tosylate at about 130° C. for 1.5 hours.

Example 6 Preparation of Compound (7)

Compound (3) was coupled with 2,6-dimethylphenylboronic acid using theconditions described in Example 3 (Preparation of compound (4)). Thiswas followed by quarternization with methyl tosylate at about 130° C.for 1.5 hours.

Example 7 Preparation of Compound (8)

Compound (3) was coupled with 4-hydroxyphenylboronic acid using theconditions described in Example 3 (Preparation of compound (4)). Thiswas followed by quarternization with methyl tosylate at about 130° C.for 1.5 hours.

Example 8 Preparation of Compound (9)

Compound (3) was coupled with benzothiaphen-2-yl boronic acid using theconditions described in Example 3 (Preparation of compound (4)). Thiswas followed by quarternization with methyl tosylate at about 130° C.for 1.5 hours.

Example 9 Preparation of Compound (10)

Compound (3) was coupled with phenylboronic acid using the conditionsdescribed in Example 3 (Preparation of compound (4)). This was followedby quarternization with methyl tosylate at about 130° C. for 1.5 hours.

Example 10 Preparation of Compound (11)

Compound (3) was coupled with thiophene-2-boronic acid using theconditions described in Example 3 (Preparation of compound (4)). Thiswas followed by quarternization with methyl tosylate at about 130° C.for 1.5 hours.

Example 11 Preparation of Compound (12)

Compound (3) was coupled with 2-acetamidobenzeneboronic acid using theconditions described in Example 3 (Preparation of compound (4)). Thiswas followed by quarternization with methyl tosylate at about 130° C.for 1.5 hours.

Example 12 Preparation of Compound (13)

To 1.5 g NaH (60% in dispersion oil and washed with hexanes) in 50 mlDMF 1.5 g of 2-hydroxyl-4-methylquinoline was added followed by 4.5 mLof benzyl bromide. The mixture was stirred at room temperature for 2hours. Solvent was removed under reduced pressure and the residue wasdissolved in CHCl₃ and washed with water. The product was obtained bysilica gel column purification with ethyl acetate and hexanes.

Example 13 Preparation of Compound (14)

To 0.5 g of Compound (5), 0.25 g of Compound (13) in 10 mL of methylenechloride, 0.7 mL of diisopropylethylamine and 0.9 mL of trimethylsilyltrifluoromethanesulfonate were added, and the resulting mixture washeated at reflux for 1 hour. The mixture was washed with water, and theproduct was purified on a silica gel column with ethyl acetate andhexanes.

Example 14 Preparation of Compound (15)

A mixture of 0.1 g of Compound (14) and 0.06 mL of phosphorousoxychloride was refluxed in 3 mL of dichloroethane for 5 hours. Themixture was washed with water and the solvent was removed. The residuewas stirred in ethyl acetate and filtered to obtain the product.

Example 15 Preparation of Compound (16)

A mixture of 0.1 g of Compound (15) and 0.3 mLN,N-dimethyl-N′-propyl-1,3-propanediamine was heated at 55° C. in 3 mLof 1,2-dichloroethane for 3 hours. The solvent was removed and theproduct was purified on a silica gel column with chloroform andmethanol.

Example 16 Preparation of Compound (17)

Compound (17) was prepared by following the same procedure used toprepare Compound (16), substituting 3,3′-iminobis(N,N-dimethylpropylamine) for N,N-dimethyl-N′-propyl-1,3-propanediamine.

Example 17 Preparation of Compound (18)

Compound (18) was prepared by following the same procedure used toprepare Compound (16), substituting N,N,N′-trimethylethanediamine forN,N-dimethyl-N′-propyl-1,3-propanediamine.

Example 18 Preparation of Compound (19)

A mixture of 12 mg of Compound (15) and 2 mg of imidazole was stirred atroom temperature in 2 mL of methylene chloride for 2 hours. At the endof the period, 2 mL of ethyl acetate was added and stirring wascontinued overnight. The product was obtained by centrifugation.

Example 19 Preparation of Compound (20)

Compound (20) was prepared by following the same procedure used toprepare Compound (16), substituting 1-methyl-piperazine forN,N-dimethyl-N′-propyl-1,3-propanediamine.

Example 20 Preparation of Compound (21)

Compound (21) was prepared by following the same procedure used toprepare Compound (16), substituting piperazine forN,N-dimethyl-N′-propyl-1,3-propanediamine.

Example 21 Preparation of Compound (22)

Compound (22) was prepared by following the same procedure used toprepare Compound (16), substituting N,N′-dimethylethanediamine forN,N-dimethyl-N′-propyl-1,3-propanediamine.

Example 22 Preparation of Compound (23)

Compound (23) was prepared by following the same procedure used toprepare Compound (16), substituting N,N′-dimethylpropanediamine forN,N-dimethyl-N′-propyl-1,3-propanediamine.

Example 23 Preparation of Compound (24)

A mixture of 18 mg of Compound (15), 5 mg of 2-N,N-dimethylaminoethanethiol hydrochloride, and 9 uL of triethylamine in 5mL of methylene chloride was stirred at room temperature for 1.5 hours.All volatile components were removed under reduced pressure and thecrude was purified on a silica gel column using chloroform and methanol.

Example 24 Preparation of Compound (25)

To 0.35 g of 4-diethylaminomethyl-bromobenzene in 4 mL dry THF at −78°C. under nitrogen, 0.32 mL of a 2.5 M n-butyllithium was introducedfollowed by 0.1 g of Compound (13) in 2 mL THF. The reaction was stirredat the low temperature for 1 hour before the addition of 1 mL aceticacid. The mixture was stirred at room temperature for another hour andthe solvent was removed and the residue was further pumped for an hour.To the residue, 0.2 g of Compound (5), 2 mL of dichloroethane and 0.35mL of triethylamine were added and stirred at room temperature for onehour. The resulting mixture was washed with dilute sodium hydroxide andpurified on a silica gel column chromatography using chloroform andmethanol.

Example 25 Preparation of Compound (26)

To 50 mg of Compound (13) in 5 mL of THF at −78° C. under nitrogen, 0.16mL of a 2.5 M n-butyllithium was added. After 30 minutes at the lowtemperature, 0.5 mL of acetic acid was added and the resulting mixturewas stirred at room temperature for 1 hour. Volatile components wereevaporated under reduced pressure and the residue further pumped invacuo for 30 minutes. To the resulting residue in 10 mL of methylenechloride, 50 mg of Compound (5) and 84 uL of triethylamine were addedand the mixture was stirred at room temperature for several hours. Theorganic layer was washed with dilute HCl and NaCl and the crude waspurified on silica gel using ethyl acetate, chloroform and methanol.

Example 26 Preparation of Compound (27)

Compound (27) was prepared by following the same procedure used toprepare Compound (15), using 4-methyl-1-phenyl-2(H)-quinolone as thestarting material.

Example 27 Preparation of Compound (28)

Compound (28) was prepared by following the same procedure used toprepare Compound (16), using Compound (27) as the starting material.

Example 28 Preparation of Compound (29)

Compound (29) was prepared by following the same procedure used toprepare Compound (15), using 1,4-dimethyl-2(H)-quinolinone as thestarting material.

Example 29 Preparation of Compound (30)

Compound (30) was prepared by following the same procedure used toprepare Compound (16), using Compound (29) as the starting material.

Example 30 Preparation of Compound (31)

A mixture of 4 mg of Compound (16) and 0.05 mL of methyl iodide in 0.5mL DMF was heated at 60° C. overnight. The solvent was removed and theproduct was purified on a LH-20 column with water.

Example 31 Preparation of Compound (32)

A mixture of 20 mg of Compound (15) and 0.05 mL triethylamine in 2 mLmethanol was heated at 60° C. for 1 day. The product was precipitatedout by the addition of ethyl acetate.

Example 32 Preparation of Compound (33)

Compound (33) was prepared by following the same procedure used toprepare Compound (32), using 2-dimethylaminoethanol instead of methanol.

Example 33 Preparation of Compound (34)

Compound (34) was prepared by following the same procedure used toprepare Compound (16) using 4-methyl-1-propargyl-2(H)-quinolinone as thestarting material to generate the intermediate 2-chloro derivative,which in turn was reacted with N,N-dimethyl-N′-propylpropanediamine togenerate the target.

Example 34 Preparation of Compound (35)

Compound (35) was prepared by following the same procedure used toprepare Compound (16), using Compound (41) as the starting material.

Example 35 Preparation of Compound (36)

To 1 g of N-benzyl-4-methoxyaniline in 10 mL of toluene was added 0.72mL of diketene. The mixture was stirred overnight and the solvent wasremoved under reduced pressure. To the residue, 8 mL of a 1:1 v/v mix ofH₂SO₄:HOAc was added and heated at 50° C. overnight. The mixture waspoured onto ice water and extracted with ethyl acetate. The crudematerial was purified on silica gel using ethyl acetate and hexanes.

Example 36 Preparation of Compound (37)

Compound (37) was prepared by following the same procedure used toprepare Compound (36), using N-benzyl-3-methoxyaniline in place ofN-benzyl-4-methoxyaniline.

Example 37 Preparation of Compound (38)

Compound (38) was prepared by following the same procedure used toprepare Compound (16), using Compound (37) as the starting material.

Example 38 Preparation of Compound (39)

Compound (38) was prepared by following the same procedure used toprepare Compound (16), using Compound (36) as the starting material.

Example 39 Preparation of Compound (40)

Compound (40) was prepared by following the same procedure used toprepare Compound (13), using bromoethylbenzene as the starting material.

Example 40 Preparation of Compound (41)

Compound (41) was prepared by following the same procedure used toprepare Compound (13), using bromoethylpyridine as the startingmaterial.

Example 41 Preparation of Compound (42)

A mixture of 0.5 g of lepidine and 2.76 g of p-xylylene dibromide wasrefluxed in 10 mL of ethyl acetate for 1 hour. The product was obtainedby filtration.

Example 42 Preparation of Compound (43)

A mixture of 0.32 g of Compound (5), 0.26 g of Compound (42), 0.4 g ofN-ethylmaleimde, and 0.16 mL of diisopropylethylamine was stirred in 5mL of methylene chloride at 0° C. for 1 hour. Next, 20 mL of ethylacetate was added, and the product was collected by filtration.

Example 43 Preparation of Compound (44)

The compound was obtained by reacting Compound (43) with an excessamount of N-methylpiperazine in DMF at room temperature for 3 hours.

Example 44 Preparation of Compound (45)

The compound was obtained by reacting Compound (43) with an excessamount of morpholine in DMF at room temperature for 3 hours.

Example 45 Preparation of Compound (46)

The compound was obtained by reacting Compound (43) with an excessamount of N,N,N′,N′-tetramethylpropanediamine in DMF at 50° C. for 4hours.

Example 46 Preparation of Compound (47)

The compound was obtained by reacting Compound (43) with an excessamount of N,N,N′-trimethylpropanediamine in DMF at 50° C. for 2 hours.

Example 47 Preparation of Compound (48)

The compound was obtained by reacting Compound (43) with an excessamount of trimethylamine in DMF at 50° C. for 4 hours.

Example 48 Preparation of Compound (49)

The compound was obtained by reacting Compound (46) with an excessamount of methyl iodide in DMF at room temperature overnight.

Example 49 Preparation of Compound (50)

The compound was obtained by reacting Compound (46) with an excessamount of 3,3′-iminobis(N,N-dimethylpropylamine) in DMF at roomtemperature for 4 hours.

Example 50 Preparation of Compound (51)

The compound was obtained by reacting Compound (46) with an excessamount of dimethylamine in DMF at 60° C. for 1 hour.

Example 51 Preparation of Compound (52)

A mixture of 0.2 g of Compound (5), 0.16 g of Compound (42), and 0.2 mLof triethylamine was stirred in 10 mL of dichloroethane at roomtemperature for 1 hour. The reaction mixture was washed with water andbrine, and the crude material was purified using HPLC with chloroformand methanol.

Example 52 Preparation of Compound (53)

A mixture of 10 mg of Compound (5), 7 mg of 1-benzyl-4-methylquinoliniumbromide, and 0.1 mL of triethylamine was stirred in 1 mL of methanol for2 hours. Volatile components were removed under reduced pressure, andthe crude was purified using silica gel chromatography with chloroformand methanol.

Example 53 Preparation of Compound (54)

A mixture of 4-bromomethylpyridine HBr and 2 equivalents of lepidine washeated at 120° C. for one hour, and followed by stirring in ethylacetate for several hours. The product was collected by filtration.

Example 54 Preparation of Compound (55)

A mixture of 10 mg of Compound (5), 5 equivalents of Compound (54) and0.1 mL of triethylamine was stirred in 0.5 mL of DMF for 1 hour.Volatile components were removed under reduced pressure, and the crudematerial was purified using silica gel chromatography with chloroformand methanol.

Example 55 Preparation of Compound (56)

A mixture of 3 mg of Compound (55) and about 0.2 mL of iodomethane wasstirred at room temperature overnight in 1 mL of DMF. Ethyl acetate (4mL) was added and after stirring for an additional hour, the product wasfiltered.

Example 56 Preparation of Compound (57)

A mixture of 1.7 mg of Compound (34), 1 mg of Cu(I)I, 50 uL ofdiisopropylethylamine, and about 5 equivalents of propylazide wasstirred at room temperature in 1 mL of methanol overnight. Volatilecomponents were removed under reduced pressure, and the crude materialwas purified using silica gel chromatography with chloroform andmethanol.

Example 57 Preparation of Compound (58)

To 0.1 g of Compound (41) in 5 mL of THF at −78° C., 0.32 mL of a 2.5 Mn-butyllithium was added and stirred at −78° C. for 1 hour. Next, 0.5 mLof acetic acid was added and stirred at room temperature for 1 hour.Volatile components were evaporated and the residue pumped in vacuo. Tothe dark residue in several mL of methylene chloride, 243 mg of Compound(5) and 0.2 mL of triethylamine were added and stirred at roomtemperature for 1 hour. The organic layer was washed with water andbrine, and dried over magnesium sulfate. The crude material was purifiedusing silica gel chromatography with ethyl acetate, chloroform andmethanol.

Example 58 Preparation of Compound (59)

A mixture of 1.26 g of 5-(2,6-dimethoxyphenyl)-2-methylbenzothiazole and0.99 g of methyl tosylate was heated at 130° C. for 1 hour. The crudematerial was stirred in about 30 mL of ethyl acetate and filtered toobtain the product.

Example 59 Preparation of Compound (60)

A mixture of 0.1 g of Compound (59), 32 mg of4-dimethylaminobenzaldehyde and 21 uL of piperidine was heated at 40° C.in 10 mL of ethanol for 1.5 hours. Volatile components were removedunder reduced pressure, and the residue was dissolved in chloroform andwashed with water and brine. The crude material was purified usingsilica gel chromatography with chloroform and methanol.

Example 60 Preparation of Compound (61)

A mixture of 1 g of lepidine and 1 mL of (2-bromoethyl)benzene washeated at 90° C. for 2 hours. About 30 mL of ethyl acetate was added andrefluxed for 15 minutes. The product was collected by filtration.

Example 61 Preparation of Compound (62)

A mixture of 0.1 g of Compound (5), 67 mg of Compound (61), and 86 uL oftriethylamine in 10 mL of dichloroethane was stirred at room temperaturefor 1 hour. Volatile components were evaporated under reduced pressure,and the residue was stirred in about 30 mL of ethyl acetate at roomtemperature overnight. The product was collected by filtration.

Example 62 Preparation of Compound (63)

A mixture of 40 uL of lepidine and 57 mg of2-(acetamido)-4-(chloromethyl)-thiazole was heated at 90° C. for 2hours. The crude material was stirred in about 20 mL of ethyl acetatefor several hours, and filtered to obtain the product.

Example 63 Preparation of Compound (64)

A mixture of 0.13 g of Compound (5), 0.26 mmole of Compound (63), and0.11 mL of triethylamine was stirred in 10 mL of dichloroethane at roomtemperature for 1 hour. The crude product was purified using silica gelchromatography with chloroform and methanol.

Example 64 Preparation of Compound (65)

A mixture of 0.1 g of Compound (5), 58 mg of 1,4-dimethylquinoliniumiodide, and 86 uL of triethylamine was stirred in 10 mL ofdichloroethane at room temperature for 1 hour. Volatile components wereevaporated under reduced pressure, and the residue was stirred in about50 mL of ethyl acetate for 30 minutes. The product was collected byfiltration.

Example 65 Preparation of Compound (66)

Compound (66) was prepared by following the same procedure used toprepare Compound (15), using Compounds (7) and (13) as the startingmaterials.

Example 66 Preparation of Compound (67)

Compound (67) was prepared by following the same procedure used toprepare Compound (15), using Compound (7) and4-methyl-1-phenyl-2(H)-quinolone as the starting materials.

Example 67 Preparation of Compound (68)

Compound (68) was prepared by following the same procedure used toprepare Compound (16), using Compound (67) andN,N-dimethyl-N′-propyl-propanediamine as the starting materials.

Example 68 Preparation of Compound (69)

Compound (69) was prepared by following the same procedure used toprepare Compound (16), using Compound (67) andN,N,N′-trimethylpropanediamine as the starting materials.

Example 69 Preparation of Compound (70)

Compound (70) was prepared by following the same procedure used toprepare Compound (16), using Compound (66) andN,N,N′-trimethylpropanediamine as the starting materials.

Example 70 Preparation of Compound (71)

A mixture of 20 mg of Compound (7), 13 mg of1-benzyl-4-methyl-quinolinium bromide, and 50 uL of triethylamine wasstirred in 1 mL of methanol at room temperature for 1 hour. Volatilecomponents were evaporated under reduced pressure, and the crudematerial was purified using silica gel column chromatography withchloroform and methanol.

Example 71 Preparation of Compound (72)

A mixture of 20 mg of Compound (7), 17 mg of Compound (42), and 50 uL oftriethylamine was stirred at room temperature in 1 mL of DMF for 1 hour.This was followed by the addition of about 100 uL of 1-methylpiperazine,and the mixture was stirred overnight. Volatile components wereevaporated under reduced pressure, and the product was purified usingsilica gel chromatography with chloroform and methanol.

Example 72 Preparation of Compound (73)

A mixture of 8 mg of Compound (7), 8 mg of Compound (42) and 0.5 mL oftrimethylamine (25% in methanol) was stirred at room temperature in 1 mLof DMF for 4 hours. Volatile components were evaporated under reducedpressure, and then pumped in vacuo. The crude material was purifiedusing silica gel chromatography with chloroform and methanol.

Example 73 Preparation of Compound (74)

A mixture of 20 mg of Compound (7), 5 equivalents of Compound (54), and50 uL of triethylamine was stirred in 1 mL of DMF at room temperaturefor 1 hour. Volatile components were evaporated under reduced pressure.The crude material was purified using silica gel column chromatographywith chloroform and methanol.

Example 74 Preparation of Compound (75)

A mixture of about 3 mg of Compound (74) and 0.2 mL of iodomethane wasstirred at room temperature overnight in 1 mL of DMF. The product wasprecipitated by addition of 4 mL of ethyl acetate.

Example 75 Preparation of Compound (76)

Compound (76) was prepared by following the same procedure used toprepare Compound (15), using Compound (6) and4-methyl-1-phenyl-2(H)-quinolone as the starting materials.

Example 76 Preparation of Compound (77)

Compound (77) was prepared by following the same procedure used toprepare Compound (16), using Compound (15) andN,N-dimethyl-N′-propylpropanediamine.

Example 77 Preparation of Compound (78)

A mixture of about 35 mg of Compound (6), 30 mg of Compound (42), and 17uL of diisopropylethylamine was stirred in 5 mL of a 1:4 v/vDMF/methylene chloride solvent at room temperature overnight. Volatilecomponents were evaporated under reduced pressure, and the crude productwas purified using silica gel column chromatography with ethyl acetate,chloroform and methanol.

Example 78 Preparation of Compound (79)

A mixture of about 5 mg of Compound (78) and 3 mL of a 2 M solution ofdimethylamine in THF was heated in 10 mL of methanol at room temperaturefor 3 days. Volatile components were removed under reduced pressure, andthe crude product was purified using silica gel column chromatographywith chloroform, methanol and triethylamine.

Example 79 Preparation of Compound (80)

A mixture of about 5 mg of Compound (78) and 30 mg of 1-methylpiperazinewas stirred at 35° C. for 4 days. Volatile components were evaporatedunder reduced pressure, and the product was purified on a preparatoryTLC plate.

Example 80 Preparation of Compound (81)

A mixture of 20 mg of Compound (6), 20 mg of1-benzyl-4-methylquinolinium bromide, and 0.1 mL triethylamine wasstirred in 0.5 mL of methylene chloride at room temperature for 1 hour.Volatile components were evaporated under reduced pressure, and thecrude material was purified using silica gel column chromatography withmethanol and chloroform.

Example 81 Preparation of Compound (82)

To 1.3 g of 1-methylimidazole in 20 mL of THF at −78° C. under nitrogen,2.8 mL of a 2.5 M n-butyllithium was introduced. After 45 minutes at thelow temperature, 1.25 g of 1-benzyl-4-methyl-2(H)-quinolone (in 10 mL ofTHF) was added and the resulting mixture was further stirred at −78° C.for 1 hour, at 0° C. for 2 hours and room temperature for another 30minutes. Acetic acid (0.5 mL) was added and stirred for 30 minutes.Volatile components were removed under reduced pressure. The resultingmaterial was presumably1-benzyl-4-methyl-2-(1-methylimidazoyl)-quinolinium acetate.

Example 82 Preparation of Compound (83)

To a mixture of 20 mg of Compound (6) and 0.01 mmole of Compound (82) in1 mL of methanol, 50 uL of triethylamine was added and stirred at roomtemperature for 1 hour. Volatile components were evaporated. The crudematerial was purified using silica gel column chromatography withchloroform and methanol, and then on a LH-20 column with water to obtainthe pure product.

Example 83 Preparation of Compound (84)

The compound was prepared from the commercially available5-phenyl-2-mercapto-benzothiazole (Aldrich Chemical; St. Louis, Mo.) byfirst converting the mercapto into a methylthio with potassium carbonateand methyl tosylate, and further quarternization of the benzothiazoleunder neat condition with methyl tosylate to generate the desiredcompound.

Example 84 Preparation of Compound (85)

A mixture of 60 mg of Compound (84), one molar equivalent of1,4-dimethyl-2-(1-methylimidazoyl)-quinolinium acetate (prepared bysimilar protocol to that of Compound (82) using1,4-dimethyl-2(H)-quinolone as the starting material), and 0.1 mL oftriethylamine was stirred in 1 mL of methanol at room temperature for 1hour. The crude material was purified on a LH-20 column eluting withwater.

Example 85 Preparation of Compound (86)

A mixture of 15 mg of Compound (10) and 9.6 mg of1,4-dimethylquinolinium iodide in 1 mL of methylene chloride was stirredat room temperature for 1 hour. The product was obtained by filtration.

Example 86 Preparation of Compound (87)

A mixture of 27 mg of Compound (10), 0.067 mmole of1,4-dimethyl-2-(1-methylimidazoyl)-quinolinium acetate (prepared bysimilar protocol to that of Compound (82) using1,4-dimethyl-2(H)-quinolone as the starting material), and 0.1 mL oftriethylamine was stirred in 1 mL of methylene chloride for 1 hour.Volatile components were evaporated, and the crude product was purifiedon a LH-20 column.

Example 87 Preparation of Compound (88)

To a mixture of 8 mg of Compound (84) and one equivalent of Compound(82) in 1 mL of methanol, 50 uL of triethylamine was added and stirredat room temperature for 1 hour. Volatile components were evaporated, andthe crude material was purified first by silica gel columnchromatography with chloroform and methanol and second on a LH-20 columnwith water to obtain the pure product.

Example 88 Preparation of Compound (89)

A mixture of 43 mg of 5-phenyl-3-methyl-2-methylthiobenzoxazoliumtosylate, 39 mg of 1-benzyl-4-methylquinolinium bromide, and 0.1 mL oftriethylamine was stirred in 1 mL of methylene chloride for 1 hour. Theproduct was collected by filtration.

Example 89 Preparation of Compound (90)

To 0.36 g of 4-diethylaminomethyl-bromobenzene in 4 mL dry THF at −78°C. under nitrogen, 0.48 mL of a 2.5 M n-butyllithium was introducedfollowed by 0.235 g of 4-methyl-1-phenyl-2(H)-quinolone (in 10 mL THF).The reaction was stirred at the low temperature for 1 hour before theaddition of 1 mL acetic acid. The mixture was stirred at roomtemperature for another hour, and the solvent was removed and theresidue was further pumped for an hour. The crude product2-(4-diethylaminomethyl)-4-methyl-1-phenylquinolinium acetate was usedwithout further purification.

Example 90 Preparation of Compound (91)

A mixture of 20 mg of Compound (10), one equivalent of Compound (90),and 50 uL of triethylamine was stirred in 1 mL of methylene chloride atroom temperature for 1 hour. Volatile components were removed, and thecrude material was purified using silica gel column chromatography withchloroform and methanol.

Example 91 Preparation of Compound (92)

A mixture of 12 mg of Compound (10), 10 mg of1-benzyl-4-methylpyridinium bromide, and 0.1 mL of triethylamine in 1 mLof methylene chloride was refluxed for two hours. Volatile componentswere removed under reduced pressure, and the crude material was stirredin about 2 mL of methylene chloride for 1 hour. The product wascollected by filtration.

Example 92 Preparation of Compound (93)

A mixture of 8 mg of Compound (10), 8.8 mg of1-benzyl-4-methyl-2-phenylquinolinium bromide, and 50 uL oftriethylamine was refluxed in 2 mL of methylene chloride for 3 hours.The crude material was purified using silica gel chromatography withchloroform and methanol.

Example 93 Preparation of Compound (94)

A mixture of 22 mg of Compound (12), 10 mg of1-benzyl-4-methylquinolinium bromide, and 50 uL of triethylamine wasstirred in 1 mL of methanol at room temperature for one hour. The crudematerial was purified using silica gel chromatography with chloroformand methanol.

Example 94 Preparation of Compound (95)

A mixture of 6.5 mg of Compound (9), 4 mg of1-benzyl-4-methylquinolinium bromide, and 50 uL of triethylamine wasstirred in 1 mL of methanol at room temperature for 3 hours. The crudematerial was purified using silica gel chromatography with chloroformand methanol.

Example 95 Preparation of Compound (96)

A mixture of 6.8 mg of Compound (11), 4.7 mg of1-benzyl-4-methylquinolinium bromide, and 50 uL of triethylamine wasstirred in 1 mL of methylene chloride at room temperature for 1 hour.The crude material was purified using silica gel column chromatographywith chloroform and methanol.

Example 96 Preparation of Compound (97)

A mixture of 18 mg of 3-methyl-6-pyridyl-1,3-benzothiazole-2-thione, 22mg of 1-benzyl-4-methylquinolinium bromide, 14 mg of methyl tosylate,and 0.1 mL of diisopropylethylamine was heated at 100° C. for 30minutes. Volatile components were removed under reduced pressure, andthe crude product was purified by preparative TLC plate.

Example 97 Preparation of Compound (98)

A mixture of 23 mg of 3-methyl-6-pyridyl-1,3-benzolthiazole-2-thione and330 mg of methyl tosylate was heated at 130° C. for 1 hour. Next, 10 mLof ethyl acetate was added and refluxed for 15 minutes. The product wascollected by filtration.

Example 98 Preparation of Compound (99)

A mixture of 27 mg of 1-benzyl-4-methylquinolinium bromide, oneequivalent of Compound (98), and 0.2 mL of triethylamine was stirred in2 mL of DMF at room temperature. The product was collected byfiltration.

Example 99 Preparation of Compound (100)

A mixture of 50 mg of Compound (8), 1.2 equivalent of1-benzyl-4-methylquinolinium bromide, and 45 uL of triethylamine wasstirred in a mixed solvent of dichloroethane/DMF (v/v, 1:1, 4 mL) atroom temperature for 3 hours. The reaction mixture was diluted withchloroform, washed with water, and dried over magnesium sulfate. Theproduct precipitated out from the chloroform later as the volume wasreduced.

Example 100 Preparation of Compound (101)

Compound (101) was prepared by following the same procedure used toprepare Compound (16), using 6-(bis-(1,3-dimethoxy)-prop-2-yl)-3-methyl-2-methylthio-benzothiazolium tosylate andCompound (13) as the starting materials.

Example 101 Preparation of Compound (102)

Compound (102) was prepared by following the same procedure used toprepare Compound (24), using Compound (5) and1-benzyl-4-methyl-pyridin-2-one as the starting materials.

Example 102 Preparation of Compound (103)

Acetic anhydride (0.1 mL) was added to a mixture of 0.127 mg of2-(2-anilinovinyl)-3-methyl-6-phenylquinolinium tosylate, one equivalentof 1-benzyl-4-methylquinolinium bromide, and 40 uL of triethylamine in 2mL of dichloroethane at room temperature. The mixture was stirred for 2hours. The reaction was diluted with chloroform and washed with waterand brine. The crude material was purified by recrystallizing frommethanol and ethyl acetate.

Example 103 Preparation of Compound (104)

Acetic anhydride (90 uL) was added to a mixture of 0.107 mg of2-(2-anilinovinyl)-3-methyl-6-phenylquinolinium tosylate, one equivalentof 1-((3-ethoxycarbonyl-1-propoxy)phenylmethyl)-4-methylquinoliniumchloride, and 40 uL of triethylamine in 5 mL of dichloroethane at roomtemperature. The mixture was stirred for 2 hours. The reaction mixturewas diluted with chloroform and washed with water and brine. The crudematerial was purified using silica gel column chromatography withchloroform and methanol.

Example 104 Preparation of Compound (105)

Water (0.5 mL) and 40 uL of 10% sodium hydroxide was added to 59 mg ofCompound (107) in 5 mL of methanol. The mixture was stirred at roomtemperature for several hours. The reaction was diluted with about 30 mLof water, acidified with 1 N HCl, and filtered to recover the product.

Example 105 Preparation of Compound (106)

O-(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (8.2mg) was added to 11.3 mg of Compound (105) in 2 mL of DMF and 8 uL oftriethylamine in 2 mL of DMF. The mixture was stirred overnight at roomtemperature. About 6 mL of ethyl acetate was added to precipitate theproduct, and the product was obtained by filtration.

Example 106 Preparation of Compound (107)

Compound (107) was prepared by following the same procedure used toprepare Compound (15), using Compound (5) and1-benzyl-6-(3-ethoxycarbonyl-1-propoxy)-4-methyl-2(H)-quinolone as thestarting materials.

Example 107 Preparation of Compound (108)

A mixture of 0.314 g of Compound (107), 0.13 mL of thiophenol, and 0.3mL of triethylamine was stirred in 5 mL of dichloroethane at 60° C. forseveral hours. The product was purified using silica gel columnchromatography with chloroform and methane.

Example 108 Preparation of Compound (109)

4-N-methylaminobutyric acid (39 mg) and 107 uL of triethylamine wasdissolved in a mixture of 1.5 mL of isopropyl alcohol and several dropsof water. This mixture was added to a solution of 30 mg of Compound (15)in 3 mL of dichloroethane and the resulting mixture was heated at 60° C.for 1 hour. The crude material was diluted with additional chloroformand washed with diluted aqueous HCl. The product was purified on asilica gel column with chloroform and methanol.

Example 109 Preparation of Compound (110)

O-(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (7.4mg) was added to a mixture of 11 mg of B36-13-LY and 5 uL oftriethylamine in 2 mL of DMF. After 30 minutes stirring at roomtemperature, the crude material was purified on a silica gel columneluting with chloroform and acetone.

Example 110 Preparation of Compound (111)

Amino-dPEG4-alcohol (3.0 mg; Quanta Biodesign, Ltd.; Powell, Ohio) wasadded to a mixture of about 5 mg of Compound (110) and 2 equivalents oftriethylamine. The mixture was stirred for 30 minutes. The mixture wasconcentrated and several mL of ethyl acetate was added and stirredbriefly and filtered to obtain the product.

Example 111 Preparation of Compound (112)

Triethylamine (61 uL) was added to a mixture of 59 mg of1-((3-ethoxycarbonyl-1-propoxy)phenylmethyl)-4-methylquinoliniumchloride and 74 mg of Compound (5) in 3 mL of methylene chloride. Themixture was stirred for 5 minutes. The mixture was then diluted withchloroform and washed with 1:1 mixture of water/brine to yield theproduct.

Example 112 Method for Determination of DNA/RNA Fluorescence Ratios

The tested compound was dissolved as a stock solution in DMSO at aconcentration of about 0.1-1.0 mg/mL. The exact concentration is notcritical. Three tubes are prepared, each containing the same 1-20 uL ofstock solution. The first tube contained 10 mM Tris, 1 mM EDTA (pH 7.5)buffer; the second tube contained buffer and 65 ug/mL calf thymus doublestranded DNA; and the third tube contained buffer and 65 ug/mL ribosomalRNA. The tubes were incubated at room temperature for 10-15 minutes withprotection from light.

Fluorescence scans of the three solutions were performed in disposablecuvettes, with the excitation wavelength corresponding to the absorptionmaximum for the compound bound to DNA (or RNA if the values weresignificantly different). In some cases, it was necessary to dilute thesample to keep the fluorescence signal on-scale. In these cases, allthree samples were diluted to the same degree. The ratio of thefluorescence of the compound in the presence of DNA and RNA wasdetermined.

Example 113 DNA/RNA Fluorescence Ratios for Prepared Compounds

The following compounds were selected as representative of the inventiveclass of compounds. The fluorescence values in the presence of DNA andRNA were determined as described in the previous Example. The followingtable shows the DNA/RNA fluorescence ratios, where higher valuesindicate a selectivity for DNA. A ratio of 1 would indicate noselectivity. Excitation and emission values are in nm.

DNA/RNA Compound ex/em (fluorescence ratio) Thiazole orange 510/530 1Compound 16 504/532 15.2 Compound 17 508/536 4.8 Compound 18 505/533 3.5Compound 19 525/553 7.3 Compound 20 510/536 4 Compound 21 510/534 5.4Compound 22 505/534 4.7 Compound 23 500/529 3.8 Compound 24 520/570 4.6Compound 25 518/559 9 Compound 26 505/541 36 Compound 28 497/527 6.5Compound 30 495/527 5.7 Compound 31 505/534 10.5 Compound 32 498/517 36Compound 34 503/532 5.8 Compound 38 497522 13 Compound 39 507/536 6.1Compound 44 510/540 22 Compound 45 511/541 13.2 Compound 46 512/542 11.5Compound 47 512/541 8.6 Compound 48 513/540 7.3 Compound 49 520/542 18Compound 50 512/541 9.7 Compound 53 510/540 60 Compound 55 513/541 10.9Compound 56 515/545 3.7 Compound 57 506/536 3.8 Compound 62 510/539 48Compound 65 506-536 11.3 Compound 68 498/523 7 Compound 69 494/523 3.2Compound 70 501/524 2.7 Compound 89 485/512 1.8 Compound 91 522/563 1.5Compound 92 458/501 1 Compound 93 466/514 1.5 Compound 94 513/530 2.1Compound 95 550/591 1.9 Compound 96 527/555 8 Compound 97 513/538 3.7Compound 99 511/534 1.3 Compound 100 513/551 8.1 Compound 101 503/527 3Compound 102 475/515 3.3 Compound 103 507/537 3.1 Compound 104 652/666 9Compound 109 496/525 121 Compound 111 497/527 64 Compound 112 500/541191

Example 114 Evaluation of Compound (24)

This compound has a 520 nm excitation maximum, and can effectively beexcited with either a 488 nm line (blue laser) or a 532 nm (green laser)line. The compound has an emission maximum of 569 nm (orange).

Example 115 Use of Compound (20) in Flow Cytometry

Live Jurkat cells (human T-lymphocyte) were suspended at 1×10⁶ cells/mlin RPMI media with 10% Fetal Bovine Serum (FBS). 5 μM Compound (20) wasadded to one mL cell suspension, and incubated at 37° C. for 60 minutesprotected from light. Cells were processed using a Becton Dickinson (BD)LSRII Flow Cytometer. A Forward Scatter (FS) vs Side Scatter (SS) dualparameter plot was used to gate main cell population. On gated cells, adual-parameter plot of Fluorescence-Width vs Fluorescence-Area was usedfor single cell discrimination gating. Single color fluorescence wascollected at 530/30 bandpass using the 488 nm excitation laser,collecting 30,000 events at flow rate of about 200 events/second. Thedata was further analyzed using ModFit LT Flow Cytometry ModelingSoftware from Verity Software House, Inc. to determine the ratio ofG2/G1 and the CV of G1 phase.

Typical cell cycle histograms were demonstrated showing G0G1 phase, Sphase, and G2M phase. This was obtained on the live cell gate. Furtheranalysis using ModFit Software showed that G1-phase is 47.08% with peakCV of 6.92%, S-phase is 46.67%, G2-phase is 6.25% and the G2/G1 ratio is1.83. This demonstrated that the compound stains live cells for cellcycle where the CV of G1-phase <8%, and the observed ratio indicatedlinearity of staining.

Example 116 Use of Compound (24) in Flow Cytometry

Live Jurkat cells were treated with colcemid for 2 hours, to arrest cellcycle at mitosis, thus resulting in a larger more defined G2M-phase. Thecells were suspended at 1×10⁶ cells/ml in RPMI media with 10% FetalBovine Serum (FBS). 10 μM Compound (24) was added to one mL cellsuspension, incubated at 37° C. for 30 minutes protected from light.Cells were processed using the Becton Dickinson (BD) LSRII FlowCytometer. Forward Scatter (FS) vs Side Scatter (SS) dual parameter plotwas used to gate main cell population. On gated cells, a dual-parameterplot of Fluorescence-Width vs Fluorescence-Area was used for single celldiscrimination gating. Single color fluorescence was collected at 585/42bandpass using the 488 nm excitation laser, and also collected at thesame 585/42 bandpass using the 532 nm excitation laser, collecting30,000 events at flow rate of about 200 events/second. The data wasfurther analyzed using ModFit LT Flow Cytometry Modeling Software fromVerity Software House, Inc. to determine the ratio of G2/G1 and the CVof G1 phase.

Typical cell cycle histograms were demonstrated showing G0G1 phase, Sphase, and G2M phase. This was obtained on the live cell gate. Furtheranalysis using ModFit Software showed typical cell cycle staining. Thisdemonstrated that the compound stains live cells for cell cycle wherethe CV of G1-phase <8%, and the observed ratio indicated linearity ofstaining.

Example 117 Use of Compound (20) in Flow Cytometry

Live HL60 cells (human promyeloblasts) were suspended at 1×10⁶ cells/mlin Iscove's Dulbecco's complete Media with 20% Fetal Bovine Serum (FBS).5 μM Compound (20) is added to one mL cell suspension, and incubated at37° C. for 30 minutes protected from light. Cells were processed usingthe Becton Dickinson (BD) LSRII Flow Cytometer. Forward Scatter (FS) vsSide Scatter (SS) dual parameter plot was used to gate main cellpopulation. On gated cells, a dual-parameter plot of Fluorescence-Widthvs Fluorescence-Area was used for single cell discrimination gating.Single color fluorescence was collected at 530/30 bandpass using the 488nm excitation laser, collecting 30,000 events at flow rate of about 200events/second. The data was further analyzed using ModFit LT FlowCytometry Modeling Software from Verity Software House, Inc. todetermine the ratio of G2/G1 and the CV of G1 phase.

Typical cell cycle histograms were demonstrated showing G0G1 phase, Sphase, and G2M phase. This was obtained on the live cell gate. Furtheranalysis using ModFit Software showed typical cell cycle staining. Thisdemonstrated that the compound stained live cells for cell cycle wherethe CV of G1-phase <8%, and the observed ratio indicated linearity ofstaining.

Example 118 Use of compound (24) in flow cytometry

HL60 cells were suspended at 1×10⁶ cells/ml in Hanks Balanced SaltSolution (HBSS). 10 μM Compound (24) was added to one mL cellsuspension, and incubated at room temperature for 30 minutes protectedfrom light. Cells were processed using the Becton Dickinson (BD) LSRIIFlow Cytometer. Forward Scatter (FS) vs Side Scatter (SS) dual parameterplot was used to gate main cell population. On gated cells, adual-parameter plot of Fluorescence-Width vs Fluorescence-Area was usedfor single cell discrimination gating. Single color fluorescence wascollected at 585/42 bandpass using the 488 nm excitation laser, and alsoat the same 585/42 bandpass using the 532 nm excitation laser,collecting 30,000 events at flow rate of about 200 events/second. Thedata was further analyzed using ModFit LT Flow Cytometry ModelingSoftware from Verity Software House, Inc. to determine the ratio ofG2/G1 and the CV of G1 phase.

Typical cell cycle histograms were demonstrated showing G0G1 phase, Sphase, and G2M phase. This was obtained on the live cell gate. Furtheranalysis using ModFit Software showed typical cell cycle staining. Thisdemonstrated that the compound stained live cells for cell cycle wherethe CV of G1-phase <8%, and the observed ratio indicated linearity ofstaining.

Example 119 Use of Compound (24) in Flow Cytometry with Fixed Cells

HL60 cells were fixed with 70% ethanol and stored at −20° C. until use.The fixed cells were washed once in Hanks Balanced Salt Solution (HBSS)and were then suspended at 1×10⁶ cells/ml in HBSS. 5 μM Compound (24)was added to one mL cell suspension, and incubated at 37° C. for 5minutes protected from light. Cells were processed using the BectonDickinson (BD) LSRII Flow Cytometer. Forward Scatter (FS) vs SideScatter (SS) dual parameter plot was used to gate main cell population.On gated cells, a dual-parameter plot of Fluorescence-Width vsFluorescence-Area was used for single cell discrimination gating. Singlecolor fluorescence was collected at 585/42 bandpass using the 488 nmexcitation laser, and also collected at the same 585/42 bandpass usingthe 532 nm excitation laser, collecting 30,000 events at flow rate ofabout 200 events/second. The data was further analyzed using ModFit LTFlow Cytometry Modeling Software from Verity Software House, Inc. todetermine the ratio of G2/G1 and the CV of G1 phase.

Typical cell cycle histograms were demonstrated showing G0G1 phase, Sphase, and G2M phase. Further analysis using ModFit Software showedtypical cell cycle staining. This demonstrated that the compound stainedlive cells for cell cycle where the CV of G1-phase <8%, and the observedratio indicated linearity of staining.

Example 120 Use of Compound (20) for Flow Cytometry with Fixed Cells

Live Jurkat cells were treated with colcemid for 2 hours, to arrest cellcycle at mitosis, thus resulting in a larger more defined G2M-phase. Thecells were fixed with 70% ethanol and stored at −20° C. until use. Thefixed cells were washed once in Hanks Balanced Salt Solution (HBSS) andwere then suspended at 1×10⁶ cells/ml in HBSS. 5 μM Compound (20) wasadded to one mL cell suspension, and incubated at 37° C. for 5 minutesprotected from light. Cells were processed using the Becton Dickinson(BD) LSRII Flow Cytometer. Forward Scatter (FS) vs Side Scatter (SS)dual parameter plot was used to gate main cell population. On gatedcells, a dual-parameter plot of Fluorescence-Width vs Fluorescence-Areawas used for single cell discrimination gating. Single colorfluorescence was collected at 530/30 bandpass using the 488 nmexcitation laser, collecting 30,000 events at flow rate of about 200events/second. The data was further analyzed using ModFit LT FlowCytometry Modeling Software from Verity Software House, Inc. todetermine the ratio of G2/G1 and the CV of G1 phase.

Typical cell cycle histograms were demonstrated showing G0G1 phase, Sphase, and G2M phase. Further analysis using ModFit Software showedtypical cell cycle staining. This demonstrated that the compound stainedlive cells for cell cycle where the CV of G1-phase <8%, and the observedratio indicated linearity of staining.

Example 121 Use of Compound (24) for Flow Cytometry with InducedApoptosis Cells

Live Jurkat cells were split into 13 samples and each cell split wassuspended in complete RMPI/10% FBS. Samples was treated with 10 μMCamptothecin in DMSO to induce apoptosis, or treated with DMSO alone toact as a control. Treatment and control time was 1, 2, 3, 4, 5, and 6hours, and a time zero point. The cell suspensions were then incubatedat 37° C./5% CO₂ for a designated time. This type of experiment issometimes called an “apoptosis time course”. Each split was washed oncein complete RPMI media/10% Fetal Bovine Serum (FBS) and resuspended at1×10⁶ cells/mL in complete RPMI media with 10% FBS. Flow tubes were madeup by adding one mL designated cell suspension to which 10 μM compound(24) was added to one mL cell suspension, incubated at 37° C. for 30minutes protected from light, and the SYTOX® Blue dead cell stain (SYTOXis a registered trademark of Molecular Probes, Inc.; Eugene, Oreg.) wasadded as a dead cell discriminator. Cells were processed using theBecton Dickinson LSRII Flow Cytometer. A SYTOX® Blue stain vs compound(24) stain plot was made and a gate was made on compound (24) stainpositive and SYTOX® Blue stain negative cells to gate out dead cells,and to ensure only live cells were analyzed. Fluorescence from theSYTOX® Blue stain was collected using 405 nm excitation laser with450/50 bandpass and compound (24) stain fluorescence was collected at585/42 bandpass using the 488 nm excitation laser as well as collectedat the same 585/42 bandpass using the 532 nm excitation laser.Collection of 30,000 events occurred at a flow rate of about 200events/second. The data was further analyzed using ModFit LT FlowCytometry Modeling Software from Verity Software House, Inc. to look atratio of G2/G1 and the CV of G1 phase and the percent sub-G0 population(apoptotic population).

Typical cell cycle histograms were demonstrated showing G0G1 phase, Sphase, and G2M phase for each control time point with no sub-G0population identified. Cell cycle histograms for the cells treated withCamptothecin demonstrated a population of cells at the sub-G0 locationwhich begin to show at 3 hours induction and continue to increasethroughout the time course at each time point afterwards. Furtheranalysis using ModFit Software with an apoptotic model, showed typicalcell cycle staining for control cells and growing sub-G0 population withthe induced cells. This demonstrated that compound (24) stains livecells for identification of a sub-G0 population in apoptotic cells whichincreases with time of induction. Similar results were obtained with 532nm excitation.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the methods described herein without departing from the conceptand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the scope and concept of the invention.

1. A process for preparing a compound of structural formula (I) or (II)

the process comprising: a) reacting a compound of structural formula(III)

with a compound of structural formula (IVa) or (IVb), respectively

wherein: X is sulfur; R¹, R², and R⁴ are hydrogen; R⁵, R⁶, R⁷, R⁸, R⁹,R¹⁰, and R¹¹ independently comprise hydrogen, alkoxy, thioalkyl,thioaryl, halogen, alkyl, alkenyl, alkynyl, aryl, or combinationsthereof; and R¹² is alkyl.
 2. The process of claim 1, wherein R⁹comprises an aryl group.
 3. The process of claim 2, wherein the arylgroup is connected directly by a covalent bond.
 4. The process of claim2, wherein the aryl group is connected indirectly by one or moremethylene groups.
 5. The process of claim 4, wherein the R⁹ is a benzylgroup.
 6. The process of claim 1, wherein R¹² is C₁-C₈ alkyl.
 7. Theprocess of claim 1, wherein R¹² is methyl.
 8. The process of claim 1,wherein each of R⁵, R⁶, R⁷, and R⁸ is hydrogen.
 9. The process of claim1, wherein one or more of R⁵, R⁶, R⁷, and R⁸ is alkoxy.
 10. The processof claim 1, wherein the compound prepared is of structural formula (I).11. The process of claim 1, wherein the compound prepared is ofstructural formula (II).